The conversion of plant biomass into biofuels by engineered microbes would allow the replacement of petroleum-derived fuels with a renewable and CO2-neutral source of liquid fuels (National Research Council (U.S.), Panel on Alternative Liquid Transportation Fuels, Liquid transportation fuels from coal and biomass: technological status, costs, and environmental impacts (National Academies Press, Washington, D.C., 2009), pp. xvii, 370 p.)). Significant advances in metabolic engineering have enabled the production of compounds that are fully compatible with existing engines and fuel transport infrastructure (J. L. Fortman et al., Trends in Biotechnology 26, 375 (July 2008); J. D. Keasling, Science 330, 1355 (Dec. 3, 2010)). Production of biofuels from abundant materials, such as lignocellulosic plant biomass and cellulosic waste, avoids many of the problems associated with current grain-based feedstocks, and could slow anthropogenic CO2 increases, provided the biomass is responsibly grown and harvested (D. Tilman et al., Science 325, 270 (Jul. 17, 2009)).
Unfortunately, lignocellulose is not yet an economically viable feedstock, in part because substantial amounts of hydrolase enzymes are needed to convert it into fermentable sugars in high yields. These enzymes are typically generated in a dedicated process, either at the biorefinery or offsite, incurring substantial capital and material expenses (National Research Council (U.S.), Panel on Alternative Liquid Transportation Fuels, Liquid transportation fuels from coal and biomass: technological status, costs, and environmental impacts (National Academies Press, Washington, D.C., 2009), pp. xvii, 370 p.)). An alternative approach, known as consolidated bioprocessing, avoids these costs by combining enzyme generation, biomass hydrolysis, and fuel production into a single process step (L. R. Lynd, P. J. Weimer, W. H. van Zyl, I. S. Pretorius, Microbiol Mol Biol Rev 66, 506 (September 2002); L. R. Lynd, W. H. van Zyl, J. E. McBride, M. Laser, Curr Opin Biotechnol 16, 577 (October 2005)) (FIG. 1A). This can be achieved either by using a consortium of microbes with biomass-degrading and biofuel-producing capabilities (T. S. Bayer et al., Journal of the American Chemical Society 131, 6508 (May 13, 2009)), or by engineering both capabilities into a single organism. There have been demonstrations of engineered yeast or bacterial species fermenting various highly refined cellulosic substrates directly into ethanol or converting a model hemicellulose substrate into biodiesel (L. R. Lynd, W. H. van Zyl, J. E. McBride, M. Laser, Curr Opin Biotechnol 16, 577 (October 2005); D. C. la Grange, R. den Haan, W. H. van Zyl, Applied Microbiology and Biotechnology 87, 1195 (July 2010); E. J. Steen et al., Nature 463, 559 (Jan. 28, 2010)). However, no organism capable of converting an unrefined plant feedstock into biofuels that have the combustion properties of petrochemical fuels has yet been reported.
Thus, a need exists for a consolidated bioprocess in which cells produce advanced biofuels directly from an input of biomass without the addition of exogenous substrates or enzymes.